Tingbin Zhang

A water soluble initiator prepared through host–guest chemical interaction for microfabrication of 3D hydrogels via two-photon polymerization

Hydrogels with a precise 3D configuration (3D hydrogels) are required for a number of biomedical applications such as tissue engineering and drug delivery. Two-photon polymerization (TPP) is an advanced method to fabricate 3D hydrogels. However, TPP of 3D hydrogels has been challenged by the lack of TPP initiators with high efficiency in aqueous medium. In this study, a water soluble TPP initiator (WI) with high fabrication efficiency was prepared by combining hydrophobic 2,7-bis(2-(4-pentaneoxy-phenyl)-vinyl)anthraquinone (N) with a C2v symmetrical structure and 2-hydroxypropyl-β-cyclodextrins through host-guest chemical interaction. Both one and two-photon optical properties of WI have been investigated. In aqueous medium, WI showed a two-photon absorption cross-section of around 200 GM at the wavelength of 780 nm which was much higher compared with those of commercial initiators. The threshold energy of TPP for the resin with WI as a photoinitiator (the molar ratio of N in resin is 0.03%) was 8.6 mW. 3D hydrogels with a woodpile microstructure were further fabricated by using an average power of 9.7 mW and a scanning speed of 30 μm s-1.

Virus-Inspired Self-Assembled Nanofibers with Aggregation-Induced Emission for Highly Efficient and Visible Gene Delivery

High-efficiency gene transfer and suitably low cytotoxicity are the main goals of gene transfection systems based on nonviral vectors. In addition, it is desirable to track the gene transfer process in order to observe and explain the mechanism. Herein, inspired by viral structures that are optimized for gene delivery, we designed a small-molecule gene vector (TR4) with aggregation-induced emission properties by capping a peptide containing four arginine residues with tetraphenylethene (TPE) and a lipophilic tail. This novel vector can self-assemble with plasmid DNA to form nanofibers in solution with low cytotoxicity, high stability, and high transfection efficiency. pDNA@TR4 complexes were able to transfect a variety of different cell lines, including stem cells. The self-assembly process induces bright fluorescence from TPE, which makes the nanofibers visible by confocal laser scanning microscopy (CLSM). This allows us for the tracking of the gene delivery process.

Polycations with excellent gene transfection ability based on PVP-g-PDMAEMA with random coil and micelle structures as non-viral gene vectors

The low transfection efficiency of polycations is still a major problem for successful gene therapy. To address this issue, in this study, hydrophilic poly(vinyl pyrrolidone)-graft-poly[2-(N,N-dimethylamino)ethyl methacrylate] (PVP-g-PDMAEMA) and amphiphilic poly(vinyl pyrrolidone)-graft-poly[2-(N,N-dimethylamino)ethyl methacrylate]-block-poly(methylmethacrylate) (PVP-g-PDMAEMA-b-PMMA) were synthesized via the atom transfer radical polymerization (ATRP) method, and their properties as gene vectors were investigated subsequently. PVP-g-PDMAEMA formed random coils in water and PVP-g-PDMAEMA-b-PMMA self-assembled into spherical core-shell micelles with a very low critical micelle concentration of only 6.3 × 10-3 mg mL-1. PVP-g-PDMAEMA-b-PMMA/pDNA polyplexes demonstrated an excellent gene transfection efficiency, which showed not only much higher gene transfection efficiency than PVP-g-PDMAEMA/pDNA polyplexes, but obviously surpassed 25k PEI at low N/P ratio around 3 on 293T cell lines. Hence, the results suggested that PVP-g-PDMAEMA-b-PMMA could be a highly efficient gene vector.

Modified bovine serum albumin as an effective charge-reversal platform for simultaneously improving the transfection efficiency and biocompatibility of polyplexes

The charge-reversal strategy is usually employed in gene delivery to facilitate the endosomal escape of gene carriers and the release of the payload into cytoplasm. However, most of the charge-reversal materials are far from perfect biocompatible materials due to the cytotoxicity of themselves or their hydrolyzed products. In this study, an excellent charge-reversal material named modified bovine serum albumin (mBSA) was prepared. The charge reversal of biocompatible mBSA is a physical process and can instantly occur, which was confirmed by zeta potential, size detection and morphological studies. The introduction of mBSA can not only reduce the zeta potential of binary complexes (pDNA-PEI) but also increase the nuclease resistance ability of the pDNA-PEI binary complexes. In addition, cell viabilities tested by MTT assay and gene transfection assay demonstrated that mBSA can reduce the cytotoxicity of pDNA-PEI polyplexes and improve their gene transfection efficiency (serum free and 10% FBS medium) both in 293T and HepG2 cells at the same time. The experimental results of cell internalization and intracellular distribution of pDNA-PEI-mBSA ternary complexes confirmed that the improvement of transfection efficiency originated from the enhancement of endosomal escape of polyplexes. Therefore, mBSA has been proven to be a perfect charge-reversal platform to simultaneously improve the transfection efficiency and biocompatibility of polyplexes.

Structural impact of graft and block copolymers based on poly(N-vinylpyrrolidone) and poly(2-dimethylaminoethyl methacrylate) in gene delivery

Cationic polymers (polycations) are promising gene vectors that are conveniently synthesized and easily modified. In order to study the relationship between structures and properties of the polycations in gene delivery, a graft copolymer called poly(N-vinylpyrrolidone)-g-poly(2-dimethylaminoethyl methacrylate) (PVP-g-PDMAEMA, i.e. PgP) and a block copolymer called PVP-b-PDMAEMA (PbP) with equal molecular weight of PDMAEMA and PVP were prepared by two advanced living radical polymerization reactions including atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) techniques. Compared with PbP, PgP could condense pDNA more effectively into polyplexes with smaller size, higher zeta potential and better stability. The transfection efficiency of PgP at a low N/P ratio of 4 : 1 was not only higher than that of PbP, but also much higher than that of the commercially available PEI as the gold standard of polycations and lipofectamine. In addition, both PgP and PbP had less BSA absorption compared with PEI, indicating that PVP could resist BSA absorption. In order to understand the mechanism behind the high transfection efficiency of PgP, cellular uptake and endosomal escape of PgP/pDNA and PbP/pDNA polyplexes were investigated. The results demonstrated that the improvement of the transfection efficiency of PgP originated from the promotion of the cellular uptake and endosome/lysosome escape. This study will provide useful information on designing effective non-viral vectors for gene delivery.

Development and Validation of a Method for Determination of Encapsulation Efficiency of CPT-11/DSPE-mPEG2000 Nanoparticles

Herein we report development and validation of the method for evaluating the encapsulation efficiency of a micelle-based nanosystem composed of irinotecan hydrochloride (CPT-11) and an amphiphilic molecule DSPEmPEG2000. The results showed that the centrifugation method can be used for separation of free drug, a critical step in measuring encapsulation efficiency, and the EE of three batches of CPT-11/DSPE-mPEG2000 micelles was 90.0% ± 1.0%. The results also indicated that the conditions used in the process have to be optimized to acquire reliable data.

Transferrin-Dressed Virus-like Ternary Nanoparticles with Aggregation-Induced Emission for Targeted Delivery and Rapid Cytosolic Release of siRNA

Viruses have evolved to be outstandingly efficient at gene delivery, but their use as vectors is limited by safety risks. Inspired by the structure of viruses, we constructed a virus-mimicking vector (denoted as TR4@siRNA@Tf NCs) with virus-like architecture and infection properties. Composed of a hydrophilic peptide, an aggregation-induced emission (AIE) luminogen, and a lipophilic tail, TR4 imitates the viral capsid and endows the vector with AIE properties as well as efficient siRNA compaction. The outer glycoprotein transferrin (Tf) mimics the viral envelope protein and endows the vector with reduced cytotoxicity as well as enhanced targeting capability. Because of the strong interaction between Tf and transferrin receptors on the cell surface, the Tf coating can accelerate the intracellular release of siRNA into the cytosol. Tf and TR4 are eventually cycled back to the cell membrane. Our results confirmed that the constructed siRNA@TR4@Tf NCs show a high siRNA silencing efficiency of 85% with significantly reduced cytotoxicity. These NCs have comparable transfection ability to natural viruses while avoiding the toxicity issues associated with typical nonviral vectors. Therefore, this proposed virus-like siRNA vector, which integrates the advantages of both viral and nonviral vectors, should find many potential applications in gene therapy.

Micelle-like luminescent nanoparticles as a visible gene delivery system with reduced toxicity

Cationic polymers have been widely used as promising non-viral gene carriers, but their undesirable toxicity is a drawback. Hydrophobic modification has been developed as an efficient strategy to overcome this disadvantage. In this study, 25 kDa polyethyleneimine (PEI), the gold standard of polycations for effective gene delivery, was modified with the hydrophobic luminogen tetraphenylethene (TPE), which shows aggregation-induced emission (AIE) and has been utilized as a luminescent probe in various applications. The modified PEI (TPEI) self-assembled into micelle-like nanoparticles (TPEI-NPs) and displayed AIE behavior in aqueous media. The TPEI-NPs exhibited bright blue fluorescence and were suitable for long-term cell imaging. Compared with PEI, TPEI-NPs showed lower cytotoxicity but the transfection efficiency was nearly high. Therefore, the modification of polycations with hydrophobic fluorescent molecules represents an advanced strategy for designing visible gene vehicles with low toxicity.

Aggregation induced emission controlled by a temperature-sensitive organic–inorganic hybrid polymer with a particular LCST

In this study, an organic–inorganic temperature-sensitive polymer encapsulating tetraphenylethene (TPE) was designed. Its fluorescence intensity is sensitive to temperature changes and reaches a maximum at normal cell temperature. Firstly, the organic–inorganic hybrid polymer, polyhedral oligomeric silsesquioxane-based-poly(N-isopropylacrylamide) (POSS-b-PNIPAM) with a particular LCST of 37.5 °C, was synthesized by atom transfer radical polymerization. POSS-b-PNIPAM self-assembled in aqueous solutions to form stable micelles and then TPE was assembled into the micelles in water. The fluorescence intensity of TPE encapsulated in POSS-b-PNIPAM can be tuned by changing the temperature. The fluorescence intensity of TPE encapsulated in POSS-b-PNIPAM reached a maximum at 37 °C, which is much higher than the TPE fluorescence intensity at the same temperature. TPE encapsulated in POSS-b-PNIPAM still showed high fluorescence intensity after entering HeLa cells at 37 °C.

Structural influence of graft and block polycations on the adsorption of BSA

Protein adsorption is considered as an important factor for the low transfection efficiency of polycations in vivo. In this study, two typical polycations of equal molecular weight with different structures were chosen to investigate their adsorption on bovine serum albumin (BSA), including the block copolymer named poly (N-vinylpyrrolidone)-b-poly (2-dimethylaminoethyl methacrylate) (PVP-b-PDMAEMA, i.e. PbP) and graft copolymer named PVP-g-PDMAEMA (PgP), respectively. Fluorescence spectroscopy was used to confirm the binding constants and binding sites between polycations and BSA in static state. The binding constants were 4.1×10(4)M(-1) vs 8.3×10(4)M(-1) and binding sites were 0.3 vs 1.1 for PbP and PgP, respectively, indicating PgP had stronger binding affinity with BSA. Surface plasmon resonance (SPR) was used to study the dynamical non-specific interaction between BSA and polycations as well as the polyplexes. The numbers of both PbP and PgP adsorbed on BSA increased with concentration of polycations increasing, and the number of PgP adsorbed on BSA is higher compared with PbP when their concentration is low. When their concentration is high, the number of PbP adsorbed on BSA is more than that of PgP. However, PgP/DNA polyplexes showed higher adsorption amount compared with PbP/DNA polyplexes at different N/P ratios.